Analysis of mitochondrial recombination in the male sterile Brassica juncea cybrid Og1 and identification of the molecular basis of fertility reversion.

KEY MESSAGE: Recombinations between the parental genomes produced a novel mitochondrial genome in the cytoplasmic male sterile Brassica juncea cybrid Og1. A mitochondrial stoichiometric shift greatly reduced the molecule containing male-sterility-inducing orf138 gene leading to reversion to male fertility. An improved, chlorosis-corrected, cytoplasmic male sterile Brassica juncea cybrid Og1 derived from Ogura cytoplasm shows frequent reversion to male fertility. To determine the nature of mitochondrial recombination in the cybrid and to uncover the molecular mechanism of male fertility reversion, we sequenced the mitochondrial genomes of Og1, its isonuclear parental lines (OgRLM and Brassica juncea RLM198) and the revertant line (Og1-rt). Assembly of Og1 mitochondrial genome gave two circular molecules, Og1a (250.999 kbp) and Og1b (96.185 kbp) sharing two large direct repeat regions capable of recombining to form a single circular molecule. Og1a contains all essential mitochondrial genes, but the male-sterility-causing orf138 was uniquely present in Og1b along with 16 other complete or partial genes already represented in Og1a. Eleven and four recombinations between the parental mitochondrial genomes produced the Og1a and the Og1b molecules, respectively. Five genes were duplicated within Og1a, of which trnfM was inherited from both the parents while the other four genes, atp4, cox1 nad4L and trnM, were inherited from RLM198. RFLP analysis revealed that orf138-containing molecules were less abundant than Og1a in the male-sterile plants while og1b bearing molecules were undetectable in the revertant line. However, orf138 transcripts were amplified in RT-PCR and were also detected in northern blots revealing that Og1b molecules are not completely lost in the revertant plants. This is the first report where the mitochondrial genome of a cybrid is compared with its actual parents. The findings are discussed in the light of previous reports on mitochondrial genome recombination in cybrids.

Transmembrane START domain proteins: in silico identification, characterization and expression analysis under stress conditions in chickpea (Cicer arietinum L.).

Steroidogenic acute regulatory related transfer (StART) proteins that are involved in transport of lipid molecules, play a myriad of functions in insects, mammals and plants. These proteins consist of a modular START domain of approximately 200 amino acids which binds and transfers the lipids. In the present study we have performed a genome-wide search for all START domain proteins in chickpea. The search identified 36 chickpea genes belonging to the START domain family. Through a phylogenetic tree reconstructed with Arabidopsis, rice, chickpea, and soybean START proteins, we were able to identify four transmembrane START (TM-START) proteins in chickpea. These four proteins are homologous to the highly conserved mammalian phosphatidylcholine transfer proteins. Multiple sequence alignment of all the transmembrane containing START proteins from Arabidopsis, rice, chickpea, and soybean revealed that the amino acid residues to which phosphatidylcholine binds in mammals, is also conserved in all these plant species, implying an important functional role and a very similar mode of action of all these proteins across dicots and monocots. This study characterizes a few of the not so well studied transmembrane START superfamily genes that may be involved in stress signaling. Expression analysis in various tissues showed that these genes are predominantly expressed in flowers and roots of chickpea. Three of the chickpea TM-START genes showed induced expression in response to drought, salt, wound and heat stress, suggesting their role in stress response.

Brassica juncea Lines with Substituted Chimeric GFP-CENH3 Give Haploid and Aneuploid Progenies on Crossing with Other Lines.

Haploids and doubled haploids are invaluable for basic genetic studies and in crop improvement. A novel method of haploid induction through genetic engineering of the Centromere Histone Protein gene, CENH3, has been demonstrated in Arabidopsis. The present study was undertaken to develop haploid inducer (HI) lines of Brassica juncea based on the principles elaborated in Arabidopsis. B. juncea was found to carry three copies of CENH3 which generated five different transcripts, of which three transcripts resulted from alternative splicing. Unlike Arabidopsis thaliana where native CENH3 gene was knocked out for constructing HI lines, we used RNAi approach to knockdown the native CENH3 genes. Further, to rescue CENH3 silenced cells, a GFP-CENH3-tailswap construct having N terminal GFP fused to H3.3 tail sequences and synthetic CENH3 histone fold domain sequences was devised. A total 38 transgenic B. juncea plants were regenerated following co-transformation with both silencing and rescue cassettes and transgenics carrying either or both the constructs were obtained. Transgenic status was confirmed through PCR, Southern and qRT-PCR analyses. Co-transformed lines were crossed to untransformed B. juncea or a line expressing only GFP-tailswap. FACS and cytological analyses of progenies revealed partial or complete elimination of B. juncea chromosomes thereby giving rise to aneuploids and haploid. This is the first report in a polyploid crop demonstrating that CENH3 engineering could be used to develop HI lines.

Cytoplasmic male sterility in Brassicaceae crops.

Brassicaceae crops display strong hybrid vigor, and have long been subject to F1 hybrid breeding. Because the most reliable system of F1 seed production is based on cytoplasmic male sterility (CMS), various types of CMS have been developed and adopted in practice to breed Brassicaceae oil seed and vegetable crops. CMS is a maternally inherited trait encoded in the mitochondrial genome, and the male sterile phenotype arises as a result of interaction of a mitochondrial CMS gene and a nuclear fertility restoring (Rf) gene. Therefore, CMS has been intensively investigated for gaining basic insights into molecular aspects of nuclear-mitochondrial genome interactions and for practical applications in plant breeding. Several CMS genes have been identified by molecular genetic studies, including Ogura CMS from Japanese radish, which is the most extensively studied and most widely used. In this review, we discuss Ogura CMS, and other CMS systems, and the causal mitochondrial genes for CMS. Studies on nuclear Rf genes and the cytoplasmic effects of alien cytoplasm on general crop performance are also reviewed. Finally, some of the unresolved questions about CMS are highlighted.

An evolutionarily conserved mitochondrial orf108 is associated with cytoplasmic male sterility in different alloplasmic lines of Brassica juncea and induces male sterility in transgenic Arabidopsis thaliana.

Nuclear-mitochondrial gene interactions governing cytoplasmic male sterility (CMS) in angiosperms have been found to be unique to each system. Fertility restoration of three diverse alloplasmic CMS lines of Brassica juncea by a line carrying the fertility-restorer gene introgressed from Moricandia arvensis prompted this investigation to examine the molecular basis of CMS in these lines. Since previous studies had found altered atpA transcription associated with CMS in these lines, the atpA genes and transcripts of CMS, fertility-restored, and euplasmic lines were cloned and compared. atpA coding and downstream sequences were conserved among CMS and euplasmic lines but major differences were found in the 5' flanking sequences of atpA. A unique open reading frame (ORF), orf108, co-transcribed with atpA, was found in male sterile flowers of CMS lines carrying mitochondrial genomes of Diplotaxis berthautii, D. catholica, or D. erucoides. In presence of the restorer gene, the bicistronic orf108-atpA transcript was cleaved within orf108 to yield a monocistronic atpA transcript. Transgenic expression of orf108 with anther-specific Atprx18 promoter in Arabidopsis thaliana gave 50% pollen sterility, indicating that Orf108 is lethal at the gametophytic stage. Further, lack of transmission of orf108 to the progeny showed for the first time that mitochondrial ORFs could also cause female sterility. orf108 was found to be widely distributed among wild relatives of Brassica, indicating its ancient origin. This is the first report that shows that CMS lines of different origin and morphology could share common molecular basis. The gametic lethality of Orf108 offers a novel opportunity for transgene containment.

Association of AFLP and SSR markers with agronomic and fibre quality traits in Gossypium hirsutum L.

Molecular markers linked to QTL contributing to agronomic and fibre quality traits would be useful for cotton improvement. We have attempted to tag yield and fibre quality traits with AFLP and SSR markers using F(2) and F(3) populations of a cross between two Gossypium hirsutum varieties, PS56-4 and RS2013. Out of 50 AFLP primer combinations and 177 SSR primer pairs tested, 32 AFLP and four SSR primers were chosen for genotyping F(2) individuals. Marker-trait associations were studied for eight agronomic and five fibre quality traits through simple and multiple regression analysis (MRA) using a set of 92 AFLP polymorphic loci and four SSR markers. Simple linear regression analysis (SLRA) identified 23 markers for eight different traits whereas multiple regression analysis identified 30 markers for at least one of the 13 traits. SSR marker BNL 3502 was consistently identified to be associated with fibre strength. While all the markers identified in SLRA were also detected in MRA, as many as 16 of the 30 markers were identified to be associated with respective traits in both F2 and F3 generations. The markers explained up to 41 per cent of phenotypic variation for individual traits. A number of markers were found to be associated with multiple traits suggesting clustering of QTLs for fibre quality traits in cotton.

A novel orf108 co-transcribed with the atpA gene is associated with cytoplasmic male sterility in Brassica juncea carrying Moricandia arvensis cytoplasm.

Mitochondrial atpA transcripts were examined in cytoplasmic male sterile (CMS) and fertility restorer lines of CMS (Moricandia arvensis) Brassica juncea. Male sterile flowers had longer atpA transcripts than male fertiles. The mitochondrial atpA region of the CMS line was cloned and sequenced. The 5' and 3' ends of the atpA transcripts of the CMS and the fertility restorer lines were mapped and full-length transcripts were cloned and sequenced. A novel orf108 (open reading frame 108) co-transcribed with the atpA gene was found in the male sterile flowers. In the fertility restorer line, the transcript was cleaved within orf108 to yield monocistronic atpA transcripts.